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| United States Patent |
5,310,665
|
|
Lambeir
, et al.
|
May 10, 1994
|
Glucose isomerases having altered substrate specificity
Abstract
A method for selecting amino acid residues is disclosed which upon
replacement will give rise to an enzyme with an altered substrate
specificity. New mutant glucose isomerases with an altered substrate
specificity are provided according to this method. These altered
properties are useful in starch degradation and in other sugar conversion
reactions.
| Inventors:
|
Lambeir; Anne-Marie (Heverlee, BE);
Lasters; Ignace (Antwerpen, BE);
Quax; Wilhemus J. (Voorschoten, NL);
Van der Laan; Jan M. (Groningen, NL)
|
| Assignee:
|
Gist-brocades, N.V. (Delft, NL)
|
| Appl. No.:
|
637870 |
| Filed:
|
January 4, 1991 |
Foreign Application Priority Data
| Jan 04, 1990[EP] | 90200029.8 |
| Current U.S. Class: |
435/94; 435/234 |
| Intern'l Class: |
C12P 019/24; C12N 009/92 |
| Field of Search: |
435/234,233,94
|
References Cited
U.S. Patent Documents
| 4894331 | Jan., 1990 | Ratzkin | 435/94.
|
| 5041378 | Aug., 1991 | Drummond et al. | 435/234.
|
| Foreign Patent Documents |
| 0351029 | Jan., 1990 | EP.
| |
| WO88/08028 | Oct., 1988 | WO.
| |
| WO89/01520 | Feb., 1989 | WO.
| |
| WO90/00196 | Jan., 1990 | WO.
| |
Other References
Chen, Process Biochemistry (Jun./Jul. 1980) pp. 30-35.
Chen, Process Biochemistry (Aug./Sep. 1980) pp. 36-41.
Jeffries, Trends in Biotechnol. (1985) 3:208-212.
Wells et al., Proc. Natl. Acad. Sci. (1987) 84:5167-5171.
Wilks et al., Science (1988) 242:1541-1544.
Lindberg et al., Nature (1989) 339:632-634.
Amore et al., Nucl. Acids. Res. (1989) 17(18):7515.
|
Primary Examiner: Patterson, Jr.; Charles L.
Attorney, Agent or Firm: Morrison & Foerster
Claims
We claim:
1. A glucose isomerase which is a modified form of a naturally occurring
glucose isomerase having a determinable three dimensional structural
wherein said modification comprises substituting a different amino acid
from that occurring at at least one position of said naturally occurring
glucose isomerase, wherein said position is identified by:
selecting all residues and crystallographically assigned water molecules as
they exist in said three-dimensional structure which have at least one
atom within a sphere of 4 angstroms surrounding the atoms of a substrate
bound in the active site of said glucose isomerase and residues which are
in van der Waal's contact with said residues and water molecules thus
selected, to obtain ia first list of residues and water molecules;
discarding from said list of residues and water molecules those that are
involved in catalysis, cofactor binding and essential intersubunit
interactions to obtain a reduced list of residues and water molecules;
discarding from said reduced list of residues and water molecules those
that interfere with the structural roles of those resides discarded from
said first list to obtain a twice-reduced list of residues and water
molecules,
wherein said position to be substituted is selected from among those
residues on said twice-reduced list.
2. The modified glucose isomerase of claim 1 which has a V.sub.max for
glucose, measured at temperatures between 20.degree. C. and 85.degree. C.,
higher than that of the corresponding naturally-occurring glucose
isomerase.
3. The modified glucose isomerase of claim 1 which has a K.sub.m for
glucose, measured at temperatures between 20.degree. C. and 85.degree. C.,
lower than that of the corresponding naturally-occurring glucose
isomerase.
4. The modified glucose isomerase of claim 1 which has a V.sub.max for
xylose, measured at temperatures between 20.degree. C. and 85.degree. C.,
higher than that of the corresponding naturally-occurring glucose
isomerase.
5. The modified glucose isomerase of claim 1 which has a K.sub.m for
xylose, measured at temperatures between 20.degree. C. and 85.degree. C.,
lower than that of the corresponding naturally-occurring glucose
isomerase.
6. The modified glucose isomerase of claim 1 which has a V.sub.max
glucose/V.sub.max xylose ratio higher than that of the corresponding
naturally-occurring glucose isomerase.
7. The modified glucose isomerase of claim 1 which has a K.sub.m
xylose/K.sub.m glucose ratio higher than that of the corresponding
naturally-occurring glucose isomerase.
8. The modified glucose isomerase of claim 1 wherein the corresponding
naturally-occurring glucose isomerase is obtainable from a microorganism
of the order Actinomycetales.
9. The modified glucose isomerase of claim 8 wherein said
naturally-occurring glucose isomerase is derived from Actinoplanes
missouriensis.
10. The modified glucose isomerase of claim 1 wherein said replaced amino
acid residue is within a sphere of 4 angstroms around the oxygen atoms of
a sugar substrate coupled to the active site.
11. The modified glucose isomerase of claim 9 wherein said replaced amino
acid is selected from the group consisting of: 15Leu, 17Thr, 20Trp, 25Ala,
27Gly, 52Thr, 53Phe, 55Asp, 57Asp, 87Pro, 88Met, 89Val, 90Thr, 91Thr,
92Asn, 93Leu, 95Thr, 133Thr, 134Leu, 135Val, 136Leu, 138Gly, 140Arg,
179Ala, 180Ile, 182Pro, 186Glu, 215Asn, 216Pro, 218Thr, 219Gly, 243His,
244Ile, 246Leu, 253Lys, 254Phe, 256Gln, 257Asp, 258Leu, 290His, 291Phe,
293Tyr, 294Lys, and 295Pro.
12. The modified glucose isomerase of claim 11 wherein said replacement of
at least one amino acid is selected from the group consisting of: A25K,
M88S, T90S, V135Q, V135T, E186D, E186Q, N215S, H243N, L258K, H290H, K294Q,
and K294R.
13. The modified glucose isomerase of claim 1 which exhibits altered water
molecule configuration in a sphere of 4 angstroms around the oxygen atoms
of a sugar substrate coupled to the active site.
14. A process for producing the modified glucose isomerase of claim 1 which
comprises mutating a DNA sequence encoding a wildtype glucose isomerase at
selected nucleotide positions;
cloning the mutated sequence into an expression vector in such a manner
that the DNA sequence can be expressed;
transforming a host organism or cell with the vector;
culturing the host organism or cell; and
isolating the modified glucose isomerase from the culture.
15. A process for producing the modified glucose isomerase of claim 1 which
comprises culturing a host organism or cell transformed with a mutated
glucose isomerase DNA cloned into an expression vector in such a manner
that the DNA sequence can be expressed under conditions which favor
expression and
isolating the modified glucose isomerase from the culture.
16. A method to conduct an enzymatic conversion catalyzed by the glucose
isomerase enzyme of claim 1, which method comprises contacting a substrate
for said enzyme with said enzyme for a time period and under conditions
wherein said conversion occurs.
Description
TECHNICAL FIELD
The present invention relates to the application of protein engineering
technology to improve the properties of enzymes. Specifically, the present
invention discloses a method for selecting amino acids which upon
substitution give rise to an altered substrate specificity. The method is
applied to glucose isomerases. In other aspect the invention provides
glucose isomerases with an altered substrate specificity. These new
glucose isomerases can advantageously be used in industrial processes, for
example in the production of high fructose corn syrups (HFCS).
BACKGROUND OF THE INVENTION
Glucose isomerases catalyze the reversible isomerization of glucose to
fructose. Fructose is nowadays commonly applied as sugar substitute due to
its higher sweetness compared to e.g. sucrose and glucose.
Many microorganisms are known to produce glucose isomerase, see for example
the review articles by Wen-Pin Chen in Process Biochemistry, 15 June/July
(1980) 30-41 and August/September (1980) 36-41, in which a large number of
microorganisms, capable of producing glucose isomerase, are listed.
Several microorganisms can be used for the industrial production of glucose
isomerases, among these Streptomyces Ampullariella and Actinoplanes are
well known. The Wen-Pin Chen reference describes culture conditions for
the microorganisms and recovery and purification methods for the produced
glucose isomerases.
Generally the naturally occurring glucose isomerases also show a high
affinity for sugars other than glucose. In this respect D-xylose,
D-ribose, L-arabinose, D-allose and 6-deoxyglucose were found to be
substrates of this enzyme. The K.sub.m values of D-glucose, D-xylose and
D-ribose, were shown to vary from microorganism to microorganism and were
reported to be in the range, of 0.086-0.920, 0.005-0.093 and 0.35-0.65M,
respectively.
The K.sub.m values for xylose are significantly lower than for glucose,
which implies that the correct name for the enzyme is in fact xylose
isomerase. Furthermore, the V.sub.max of the commonly used glucose
isomerases is higher on xylose than on glucose, which also suggest that
xylose isomerase is a better name.
Since glucose isomerase is active on different substrates it may be
advantageous to alter the substrate specificity depending on the desired
reaction product, the specific process in which it is used or the wish to
avoid unwanted side-products.
For the application of glucose isomerase in HFCS production a higher
V.sub.max and a lower K.sub.m on glucose would be useful properties, since
the reaction time and the enzyme costs would be reduced.
Another application of glucose isomerase is in the conversion of xylose to
ethanol (Jeffries, T. W., Trends Biotechnol. 3 (1985) 208). A higher
activity (V.sub.max) on xylose and/or a better affinity for xylose would
be useful properties for this application.
The digestibility and the taste of feed for monogastric animals can be
improved if glucose is converted enzymatically into fructose. In practice
the application of glucose isomerase is hampered by the xylose
isomerisation activity, which causes an unwanted formation of xylulose in
feed. A glucose isomerase with no or reduced specificity (V.sub.max or
K.sub.m) for xylose would be preferred for application in feed
pretreatment.
Clearly, there is a need for altering the substrate specificity of glucose
isomerases which would at the same time widen the field of the application
of this enzyme.
Recently redesigning of the specific activity of enzymes with the aid of
protein engineering techniques has been described. Wells et al. (Proc.
Natl. Acad. Sci. U.S.A. 84 (1987) 5167) show an example for subtilisin.
Bacillus licheniformis and B. amyloliquefaciens serine proteases differ by
31% (86 amino acid residues) in protein sequence and by a factor of 60 in
catalytic efficiency on certain substrates. By substituting 3 of the 86
different amino acids from the B. amyloliquefaciens sequence by the
corresponding B. licheniformis residues the catalytic activity of the
mutant enzyme was improved nearly 60 fold.
In another paper it is described how a lactate dehydrogenase was changed
into a malate dehydrogenase by mutating glutamine 102 into arginine 102
(Wilks et al., Science 242 (1988) 1541).
In both cases referred to above, serine protease and lactate dehydrogenase,
the modification proposal was based on the comparison of the molecule to
be modified and naturally occurring enzymes, which already showed the
desired substrate specificity. In the same way the specificity of
cytochrome p450.sub.15.alpha. was changed into the specificity of
cytochrome p450.sub.coh by replacing Leu209 with Phe209 (Lindberg and
Negishi, Nature 339 (1989) 632).
For glucose isomerase no naturally occurring enzyme is known, which shows a
better specificity for glucose than for xylose. The above-mentioned
method, based on the comparison of active sites of homologous enzymes
having a different substrate specificity, can therefore not be applied to
glucose isomerase.
WO 89/01520 (Cetus) lists a number of muteins of the xylose isomerase which
may be obtained from Streptomyces rubiginosus and that may have an
increased stability. The selection of possible sites that may be mutated
is based on criteria differing from the ones used in the present
invention. More than 300 mutants are listed but no data are presented
concerning the characteristics and the alterations therein of the mutant
enzyme molecules.
SUMMARY OF THE INVENTION
The present invention discloses a method for selecting amino acids which
upon replacement lead to an altered substrate specificity of a given
enzyme. This method, which is generally applicable, is used to alter the
substrate specificity of glucose isomerase.
Thus, the invention also provides glucose isomerases with altered substrate
specificities. This altered substrate specificity is expressed in terms of
an altered substrate binding capacity and/or an altered catalytic activity
Specifically, mutant glucose isomerases are provided with both an absolute
and a relative change in substrate binding capacity and catalytic activity
on glucose and xylose as substrates.
Furthermore, mutant glucose isomerases are provided which show that
parameters, such as, increased stability (expressed in terms of decay
constant) and altered substrate specificity can be combined into a single
molecule by adding the respective mutations that have these effects.
The mutant glucose isomerases are obtained by the expression of a gene
encoding said glucose isomerase enzyme having an amino acid sequence which
differs at least in one amino acid from the wildtype glucose isomerase
enzyme.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a schematic representation of the active site of glucose
isomerase from Actinoplanes missouriensis, derived from the three
dimensional structure of the glucose isomerase - xylitol complex. The
inhibitor is shown in full detail in the centre of the figure. For the
amino acid residues only those atoms are drawn which are involved in
hydrogen bonding. Residue names are in boxes drawn with solid lines,
solvent molecules in boxes drawn with dashed lines. Metal binding sites
are represented by ovals numbered 395 and 580. Dashed lines indicate
electrostatic interactions: the thin dotted lines represent hydrogen
bonds, the fat dashed lines the proposed ligation of the metals. Strictly
conserved residues are marked by an asterix. For non-conserved residues
the substitutions found in nature are indicated.
FIGS. 2A and 2B show the alignment of the amino acid sequences of glucose
isomerases obtained from different microorganisms. The complete sequence
of Actinoplanes missouriensis glucose isomerase is given. The amino acid
sequence of Ampullariella glucose isomerase differs from that of the
published sequences (Saari, J. Bacteriol., 169, (1987) 612) by one
residue: Proline 177 in the published sequence was found to be Arginine.
The Streptomyces thermovulgaris sequence has only been established up to
amino acid 351. Undetermined residues are left blank. A dot indicates the
absence of an amino acid residue at this position as compared to any of
the other sequences. The different species are indicated by the following
symbols:
______________________________________
Ami. Actinoplanes missouriensis DSM 4643 (SEQ. ID. NO: 1)
Amp. Ampullariella species ATCC 31351 (SEQ. ID. NO: 2)
Svi. Streptomyces violaceoruber LMG 7183 (SEQ. ID. NO: 3)
Smu. Streptomyces murinus (SEQ. ID. NO: 4)
Sth. Streptomyces thermovulgaris DSM 40444
(SEQ. ID. NO: 5)
Art. Arthrobacter species (SEQ. ID. NO: 6)
Bsu. Bacillus subtilis (SEQ. ID. NO: 7)
Eco. Escherichia coli (SEQ. ID. NO: 8)
Lxy. Lactobacillus xylosus (SEQ. ID. NO: 9)
______________________________________
The secondary structure assignment was made in the structure of
Actinoplanes missouriensis. Helices in the barrel are enclosed by solid
lines. Shaded boxes indicate .beta.-strands.
FIG. 3 shows a comparison of the H290N mutant (bold) with the
wildtype-sorbitol-Mg structure
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a method for selecting amino acids, in an
enzyme, that upon substitution give rise to an altered substrate
specificity. To obtain such a mutant enzyme the DNA sequence encoding this
enzyme is altered in such a way that the encoded enzyme has one or more
selected amino acid replacements. The altered DNA is expressed in a
desired host/vector system. To select appropriate (point) mutations a
rational approach is taken, relying on the well coordinated application of
protein crystallography; molecular modelling and computational methods,
enzymology and kinetics, molecular biology and protein chemistry
techniques. The strategies for the identification of target amino acid
sites are innovative in a sense that it is recognized that point mutations
rarely cause local perturbations, but affect several different properties
of the protein structure at once, causing a change in different properties
as well. Therefore, although the described strategies make use of well
established structure-function relationships, they also provide a rational
way to avoid or correct unwanted alterations of kinetic and structural
properties.
Alteration of Substrate Specificity by Replacement of Appropriate Amino
Acids
To modulate the substrate binding to the active site of an enzyme, the
following general rules can be applied in sequential order, provided that
data are available concerning the Three-Dimensional (3D) structure for the
enzyme-substrate or enzyme-substrate-analogue/inhibitor complexes:
a) select all residues and crystallographically assigned water molecules
which have at least one atom within a sphere of 4.ANG. surrounding the
atoms of the substrate or of a substrate analogue/inhibitor bond in the
active site;
b) select all the residues which are in Van der Waals contact with the
residues and water molecules obtained by application of criterion a);
c) discard from the recorded list of residues and water molecules those
that are implied in catalysis, cofactor binding (such as metal ions and
nucleotides) and essential intersubunit interactions (in the case of
oligomeric enzymes);
d) discard those residues and water molecules that interfere with the
structural role of the above-selected residues. Model building and
analysis of the conserved nature of the target residues can be used to
identify an essential structural role;
e) to modulate the substrate affinity, one or more of the above-selected
residues can be substituted by alteration of the genetic code to change
one or more of the following interactions and properties:
e1) steric hindrance through altering the residue size;
e2) hydrophobicity/polarity of the substrate surroundings;
e3) solvation of the substrate surroundings either by providing side chains
that solvate via hydrogen bonding to groups within the substrate or
through substitutions that alter the water distribution within the
surroundings of the substrate;
e4) flexibility of individual residues, segments or the overall protein
structure by substitutions that disrupt hydrogen bonding networks,
decrease the local packing density in the surroundings of the substrate or
introduce cavities.
The above set of rules is a general one which can in principle be applied
to all enzymes provided that enough structural data are available. In
order to demonstrate the feasibility of this set of rules a specific
example will be discussed below.
Application of the General Rules to Modulate the Substrate Specificity of
Glucose Isomerase
Although the selection of residues by applying criteria a) through e) as
given above will be demonstrated here using the specific example of
Actinoplanes missouriensis glucose isomerase, it is clear that due to
extensive homology similar substitution sites can be selected in glucose
isomerases obtained from other species. The mentioned sequence homology is
demonstrated in FIG. 2 which gives an alignment of glucose isomerases
obtained from; Actinoplanes missouriensis, Ampullariella species,
Streptomyces violaceoruber, Streptomyces murinus, Streptomyces
thermovulgaris, Arthrobacter species, Bacillus subtilis, Escherichia coli
and Lactobacillus xylosus. The approach described above would also give
rise, after amino acid replacement at corresponding positions in the
glucose isomerases from the other species, to an altered substrate
specificity of other glucose isomerases.
In general, it can be assumed that where the overall homology is greater
than 65%, preferably greater than 74% (minimal homology between
Actinoplanes missouriensis and Streptomyces glucose isomerase, according
to Amore and Hollenberg, Nucl. Acids Res. 17, 7515 (1989)), and more
preferably greater than 85% and where the 3D structure is similar, amino
acid replacements will lead to similar changes in substrate specificity.
With similar changes in substrate specificity we mean the direction in
which the kinetic parameters change and not the magnitude. Specifically
one expects the glucose isomerases from species belonging to the order of
the Actinomycetales to have such a high degree of similarity that the
alteration of substrate specificities due to amino acid replacements at
the selected sites are similar. Actinoplanes missouriensis is the
preferred source of glucose isomerase to mutate.
Changes in substrate specificity according to the present invention include
all combinations of increase and decrease of V.sub.max and K.sub.m for
both glucose and xylose. A person skilled in the art will understand that
this encompasses the changes in other kinetic parameters. Furthermore, the
specificities for other substrates will inherently be changed also. The
proposed rules for changing the substrate specificity are not restricted
to the mentioned substrates, they can be applied to other substrates.
Among these are, D-ribose, L-arabinose, D-allose and 6-deoxyglucose.
Thus apart from providing a general method for altering the substrate
specificity the present invention applies this method to glucose
isomerase.
The selected amino acid replacements can be engineered in the DNA encoding
the glucose isomerase by methods well known to a person skilled in the art
(e.g. site-directed mutagenesis). The DNA encoding the glucose isomerase
or its mutants may be cloned on an expression vector and this construct
may be transformed to a host wherein the gene is expressed, the mRNA
translated and preferably the mature protein, or a precursor, secreted
from the cell. Subsequently the protein can be purified. Standard
procedures can be found in Maniatis et al. (Cold Spring Harbor, 1st and
2nd edition, 1982 and 1989 respectively).
The application of these methods gives rise to mutant glucose isomerase
enzymes, obtained by the expression of a gene encoding said glucose
isomerase enzyme having an amino acid sequence which differs at least in
one amino acid from the wildtype glucose isomerase enzyme, which mutant
glucose isomerase enzyme is characterized in that it exhibits an altered
substrate specificity. Instead of single mutants also double mutants may
be obtained. Some of these double mutants, aimed at combining the desired
properties, show that the properties are at least partially cumulative.
Examples are provided, in the present application, of mutants with both an
increased specificity for glucose and an increased stability.
The substrate specificity of the new enzymes can be tested on the substrate
that is necessary for the desired application. Here special attention is
paid to the kinetic parameters concerning glucose and xylose as a
substrate and also to the relative changes in these parameters.
FIG. 1 shows a schematic map of residues around the active site of A.
missouriensis. In the following methods are described to design mutants
which have a higher relative specificity for glucose compared to the
wildtype enzyme.
Applying the rules given above to glucose isomerase from Actinoplanes
missouriensis and using the coordinates of the sorbitol-GI-Co.sup.2+
structure we obtain the following list of residues:
Residues and waters selected by criterion (a): 16Trp, 54His, 88Met, 90Thr,
94Phe, 135Val, 137Trp, 181Glu, 183Lys, 217Glu, 220His, 245Asp, 255Asp,
292Asp, 294Lys, 26Phe and 4 water molecules that are arbitrarily denoted
as 491, 492, 690 and 887.
Additional residues were selected by criterion (b). In this case all
residues within a sphere of 2.ANG. around the above mentioned residues
(criterion (a)) were taken. Giving the following amino acids:
15Leu, 17Thr, 20Trp, 25Ala, 27Gly, 52Thr, 53Phe, 55Asp, 57Asp, 87Pro,
89Val, 91Thr, 92Asn, 93Leu, 95Thr, 133Thr, 134Leu, 136Leu, 138Gly, 140Arg,
179Ala, 180Ile, 182Pro, 184Pro, 186Glu, 215Asn, 216Pro, 218Thr, 219Gly,
221Glu, 243His, 244Ile, 246Leu, 254Phe, 256Gln, 257Asp, 290His, 291Phe,
293Tyr and 295Pro.
The catalytic residues: 54His, 183Lys, 220His; the cation ligands: 181Glu,
217Glu, 221Glu, 245Asp, 255Asp, 292Asp, water 690 and the interface
residue: 26Phe; are discarded by applying criterion (c).
Residues 16Trp, 94Phe and 137Trp form a conserved hydrophobic cluster
determining the overall shape of the substrate binding pocket and they are
therefore discarded by applying criterion (d). Furthermore residue 184Pro
is discarded by criterion (d).
The preferred residues to be substituted resulting after application of the
above mentioned criteria are shown in Table 1.
TABLE 1
______________________________________
15Leu 17Thr 20Trp 25Ala 27Gly
52Thr 53Phe 55Asp 57Asp 87Pro
88Met 89Val 90Thr 91Thr 92Asn
93Leu 95Thr 133Thr 134Leu 135Val
136Leu 138Gly 140Arg 179Ala 180Ile
182Pro 186Glu 215Asn 216Pro 218Thr
219Gly 243His 244Ile 246Leu 254Phe
256Gln 257Asp 290His 291Phe 293Tyr
294Lys 295Pro
______________________________________
Single substitutions at these sites and/or combinations of substitutions at
the different sites may lead to an improved activity either through relief
of steric hindrance or by the modulation of polarity of the sugar
environment. Different properties such as modulating the affinity and/or
catalytic efficiency for various substrates may be altered in this way.
In the specific case of increasing the specificity for glucose relative to
xylose it is important to note that the substrate specificity for wildtype
glucose isomerase decreases going from xylose to 6-deoxy-glucose to
glucose. The inhibition constant for sorbitol is larger than for xylitol.
This observation suggests that steric hindrance upon binding of the larger
substrate or inhibitor contributes to the lower specificity. In the
crystal structure of the enzyme-sorbitol-cobalt complex from A.
missouriensis the following residues are found within 4.ANG. of the
hydroxyl group attached to the C6 of the sorbitol: 16Trp, 88Met, 135Val,
90Thr and a water molecule denoted 491. The hydrophobic side chains 16Trp,
88Met, 135Val are unable to solvate the hydroxyl group. The hydroxyl group
of the 90Thr side chain is rotated away, exposing its methyl group to the
inhibitor. The water molecule (491) bridges O6 and O4 of the sorbitol. In
the structure of the enzyme-cobalt complex determined in the absence of
substrate or inhibitor, the active site contains a number of water
molecules bound at positions roughly corresponding to the positions of the
01, 02, 03, 04 and 05 of the inhibitor in the enzyme-sorbitol-cobalt
complex. However, the environment corresponding to the 06 position is not
able to accommodate a water molecule. This provides additional evidence
that the hydroxyl group on C6 is not properly solvated.
The substitutions which have been made in the collection of target residues
(by applying criteria a-e) were aimed to increase the polarity of the
environment of the substrate hydroxyl groups, particularly of the 06, and
to increase the flexibility of the active site to accommodate the larger
substrate.
Amongst others the following single and combined point mutations have been
made, leading to the intended changes in glucose specificity.
25Ala to Lys: introduction of a positively charged residue at a distance of
6 to 8.ANG. of the 01, 02 and 03 of the substrate. Disruption of the water
structure in the interface. Displacement of 26Phe, which shapes the
hydrophobic pocket accommodating the C1 aliphatic hydrogens of the
substrate.
243His to Asn: increased flexibility due to changes in the protein-water
hydrogen bonding network in the interior of the barrel.
290His to Asn: increased flexibility due to changes in the protein-water
hydrogen bonding network in the interior of the barrel, displacement of
water.
290His to Asn combined with 253Lys to Arg: the 253Lys to Arg mutation is
introduced to increase stability or prevent glycation (EP-A-0351029) which
leads to irreversible inactivation.
88Met to Ser: increasing the polarity of the 06 binding pocket, increasing
flexibility by locally altering the packing of the protein.
88Met to Ser combined with 243His to Asn: increasing the polarity of the 06
binding pocket, increasing flexibility by locally altering the packing of
the protein.
88Met to Ser combined with 290His to Asn: increasing flexibility due to
changes in the protein-water hydrogen bonding network in the interior of
the barrel, displacement of water, increasing the polarity of the 06
binding pocket.
90Thr to Ser: altered flexibility of the side chain, increasing flexibility
by locally altering the packing of the protein, altering the environment
of a substrate bound water molecule (492).
90Thr to Ser combined with 135Val to Gln: altered flexibility of the side
chain at position 90, increasing flexibility by locally altering the
packing of the protein, altering the environment of a substrate bound
water molecule (492), replacement of hydrophobic environment by a polar
residue, possibility of a direct hydrogen bond between the glutamine and
the C6 hydroxyl group.
90Thr to Ser combined with 135Val to Gln and 215Asn to Ser: altered
flexibility, replacement of hydrophobic environment by polar residues,
possibility to remove a water molecule, possibility of a direct hydrogen
bond between the 90Ser and the C6 hydroxyl group.
135Val to Thr: replacement of hydrophobic environment by a polar residue.
135Val to Gln: replacement of hydrophobic environment by a polar residue,
possibility of a direct hydrogen bond between the glutamine and the C6
hydroxyl group.
These mutation proposals are meant to exemplify a rationale which can be
applied to create novel glucose isomerases with desired specificities.
However, it will be clear to those skilled in the art that desired mutants
can be obtained by changing target residues into amino acids differing
from the mentioned ones in a similar manner.
Replacing, Removing, Shifting Bound Water Molecules in the Active Site
From comparison of the sorbitol structure with the xylitol structure it
appears that particular water molecules may interfere with proper glucose
binding. Replacement of this water by an appropriate amino acid side
chain, or moving the position of the water by introducing a shorter side
chain at the appropriate position may cause an increase in affinity.
Glucose isomerase mutant H290N, which has an enhanced specificity for
glucose (K.sub.m =250 mM, V.sub.max =41 .mu.moles/min/mg) can serve as an
example. The removing or shifting of bound water molecules in the active
site of mutant H290N is correlated with the observed increase in activity
for glucose. Similar effects may be obtained by shifting other bound water
molecules. Water molecules which are in a sphere of 4.ANG. around atoms
03, 04, 05 or 06 of the substrate are candidates to be removed or shifted
by mutating neighbouring amino acids.
In the following examples recombinant DNA techniques are applied to
introduce point mutations in the gene cloned from Actinoplanes
missouriensis. The protein is overexpressed in E. coli, purified and
characterized in vitro and in application conditions as described
elsewhere (Van Tilburg, 1983, Thesis: "Engineering aspects of biocatalysts
in industrial starch conversion", Delftse Universitaire Pers).
EXPERIMENTAL
Cloning and Expression of the D-Glucose Isomerase Gene
D-glucose isomerase (GI) is synonymously used for Dxylose isomerase
(D-xylose) ketol-isomerase (EC 5.3.1.5), an enzyme that converts D-xylose
into D-xylulose. The D-glucose isomerase from Actinoplanes missouriensis
produced by engineered E. coli strains is designated as EcoAmi (DSM) GI.
To distinguish the Actinoplanes missouriensis gene coding for GI from the
analogous E. coli xy1A gene, the former will be designated as GI.
Methods for manipulation of DNA molecules are described in Maniatis et al.
(1982, Cold Spring Harbor Laboratory) and Ausubel et al. (1987, Current
Protocols in Molecular Biology, John Wiley & Sons Inc. New York). Cloning
and DNA sequence of the glucose isomerase gene from Actinoplanes
missouriensis DSM 43046 is described elsewhere (EP-A-0351029). The derived
amino acid sequence of GI is numbered and compared with other glucose
isomerases in FIG. 2. In the following, the numbering of amino acids
refers to FIG. 2.
Wildtype and mutant GI enzymes were produced in E. coli strain K514 as
described in EP-A-0351029. Ep-A-0351029 specifically describes most of the
techniques applied in this application and it is therefore incorporated
herein by reference.
Assay of the Enzymatic Activity of the Expression Product
The enzymatic activity of glucose isomerase was assayed as described below
(1 unit of enzymatic activity produces 1.0 micromole of product
-D-xylulose or D-fructose- per minute; therefore, specific activity -spa-
is expressed as units per mg of GI enzymes).
GI activity was assayed directly by measuring the increase in absorbance at
278 nm of xylulose produced at 35.degree. C. by isomerisation of xylose by
glucose isomerases. This assay was performed in 50 mM triethanolamine
buffer, pH 7.5, containing 10 mM MgSO.sub.4, in the presence of 0.1M
xylose. The final concentration of glucose isomerase in the assay was
.+-.0.01 mg/ml, this concentration was precisely determined, prior to
dilution in the enzymatic assay mixture, by absorption spectroscopy using
an extinction coefficient of 1.08 at 278 nm for a solution of enzyme of
1.0 mg/ml.
In the D-Sorbitol Dehydrogenase Coupled Assay, enzymatic determination of
D-xylulose was performed at 35.degree. C. as previously described
(Kersters-Hilderson et al., Enzyme Microb. Technol. 9 (1987) 145) in 50 mM
triethanolamine, pH 7.5, 10 mM MgSO.sub.4, and 0.1M xylose, in the
presence cf.+-.2.times.10.sup.-8 M D-sorbitol dehydrogenase (L-iditol NAD
oxidoreductase, EC 1.1.14), and 0.15 mM NADH. The final concentration of
glucose isomerase in this assay was .+-.2.5.times.10.sup.-3 mg/ml, this
concentration was precisely determined as described above.
With glucose as a substrate GI activity can be assayed by the measurement
of D-fructose produced during the isomerization reaction using the
cysteine-carbazole method (CCM) which is based on the reaction of
ketosugars with carbazole in acids to yield a purple product (Dische and
Borenfreund, J. Biol. Chem. 192 (1951) 583). Alternatively, the D-fructose
produced during the isomerization reaction can be determined enzymatically
using sorbitol dehydrogenase and NADH.
As a measure of specificity the quotient is V.sub.max /K.sub.m is sometimes
used (Wells et al., ibid). For mutants the V.sub.max /K.sub.m values for
xylose and for glucose were routinely calculated. Measurements for xylose
parameters are carried out at 35.degree. C., whereas glucose parameters
are determined at 60.degree. C. These temperatures are chosen for
practical reasons. To find out whether conclusions about relative
specificity are generally applicable independent of the measurement
temperature, some measurements on xylose were performed at 60.degree. C.
for both wildtype and mutant enzymes. It was found that conclusions that
could be drawn concerning steady-state kinetic parameters at 60.degree. C.
were similar to those at 35.degree. C.
EXAMPLES
Example 1: Mutants with Improved Catalytic Properties
The 3-dimensional structure of Actinoplanes missouriensis glucose isomerase
was studied to select those residues which upon change might yield an
improved substrate binding, catalytic activity or substrate specificity.
Residues directly or indirectly (via another residue or water molecule)
within 4 from 01-06 of the substrate were selected and changed by
site-directed mutagenesis with the aid of the pMa, pMc vector system
(Stanssens et al., Nucl. Acids Res. 17 (1989) 4441)
In Table 2 below the enzymatic parameters of several of these selected
mutants are shown:
In Table 2 xylose is abbreviated "xy" and glucose is abbreviated "g1".
TABLE 2
__________________________________________________________________________
In Table 2 xylose is abbreviated "xy" and glucose is
abbreviated "gl".
(.mu.mol/min/mg)
K.sub.m
K.sub.m
K.sub.m
V.sub.max
V.sub.max
V.sub.max
xy gl xy/gl
V.sub.max /K.sub.m
V.sub.max/ Km
Mutant xy gl gl/xy
(mM)
(mM)
.times.10.sup.-2
xy gl
__________________________________________________________________________
Wild Type
24.20
34.80
1.43
4.80
290 1.7 5.04 0.12
A25K 7.66
22.61
2.95
2.73
207 1.3 2.81 0.11
M88S 19.37
29.44
1.51
7.60
263 2.9 2.55 0.11
M88SH243N
13.90
24.98
1.79
11.92
387 3.1 1.17 0.06
M88SH290N
16.07
49.77
3.09
14.82
406 3.6 1.08 0.12
T90S 37.07
32.77
0.88
16.76
180 9 2.21 0.18
T90SV135Q
0.54
19.30
36 182 2822
6.4 0.00 0.01
V135Q 0.67
5.57
8.3 56.00
1120
5 0.01 0.00
V135T 10.90
33.11
3.04
19.95
678 3 0.55 0.05
E186D 8.51
37.90
4.45
7.00
736 0.9 1.22 0.05
E186Q 0.77
2.06
2.68
1.80
57 3 0.43 0.04
H243N 18.90
22.00
1.16
5.80
180 3.2 3.26 0.12
K253RH290N
26.70
49.13
1.84
12.80
331 3.9 2.09 0.15
L258K 24.23
44.17
1.82
4.44
327 1.4 5.46 0.14
H290N 24.00
41.80
1.74
9.70
250 4 2.47 0.17
K294Q 5.70
9.70
1.70
32.00
309 10 0.18 0.03
K294R 13.80
27.52
4.82
4.50
308 1.5 3.07 0.09
T90SV135Q,
0.93
39.6
42.60
360 1000
36 0.0025
0.04
N215S
__________________________________________________________________________
Conditions for determining enzymatic parameters were as follows:
V.sub.max, xylose: 35.degree. C., 10 mM Mg.sup.2 +, pH 7.5;
V.sub.max, glucose: 60.degree. C., 10 mM Mg.sup.2 +, pH 7.5;
K.sub.m, xylose: 35.degree. C., 10 mM Mg.sup.2 +, pH 7.5;
K.sub.m, glucose: 60.degree. C., 10 mM Mg.sup.2 +, pH 7.5.
Activities were measured on purified enzyme. The coupled sorbitol
dehydrogenase assay was used for xylose, the cysteine-carbazole or
discontinuous sorbitol dehydrogenase method was used for glucose.
Example 2: Glucose Isomerases with Improved Affinity for Glucose
Table 3 summarizes measured values from various mutants compared to
wildtype enzyme. It can be seen that all of these mutants have a lower
K.sub.m for glucose. This means that the binding of the substrate glucose
is enhanced. Moreover, for some of the mutants the K.sub.m for xylose has
not improved but became worse, as exemplified in Table 2. Thus, mutants
M88S, T90S, T90SV135Q, V135Q, H290N, K294Q, H243N have acquired a better
K.sub.m xylose/K.sub.m glucose ratio as compared to the wild-type enzyme.
TABLE 3
______________________________________
MUTANT K(gl) K(gl)/WT
______________________________________
E186Q 57.000 0.197
H243N 180.000 0.621
T90S 180.000 0.621
A25K 207.000 0.714
H290N 250.000 0.862
M88S 203.000 0.907
Wildtype 290.000 1.000
______________________________________
The Km for glucose is expressed in mM.
Example 3: Glucose Isomerases with Enhanced Catalytic Activity on Glucose
Glucose isomerase mutants with enhanced catalytic activity on glucose are
E186D, L258K, H290N and combined mutations with H290N. L258K was not
selected using the criteria described in the general methods as the
leucine at position 258 is about 10.ANG. away from the substrate or
inhibitor.
In Table 4,the V.sub.max relative to wildtype V.sub.max is shown. An
increase from 8.9% to 43% is shown. This will give rise to a faster
isomerisation of the preferred substrate glucose.
TABLE 4
______________________________________
MUTANT V(gl) V(gl)/WT
______________________________________
M88SH290N 49.770 1.430
K253RH290N 49.130 1.412
L258K 44.170 1.269
H290N 41.800 1.201
T90SV135QN215S 39.600 1.138
E186D 37.900 1.089
Wildtype 34.800 1.000
______________________________________
The V.sub.max for glucose is expressed in micromoles/min/mg.
Mutant H290N shows a V.sub.max for glucose of 41.80 which is significantly
better than the wildtype enzyme. Moreover, the V.sub.max xylose has not
improved. The ratio V.sub.max glucose/V.sub.max xylose of H290N has
therefore been improved, rendering this mutant more into a "true" glucose
isomerase.
Example 4: Structure of H290N Glucose Isomerase
In the crystal structure of the mutant H290N, it is observed that the amide
group of the Asn side chain is superimposable on the imidazole group of
w.t. molecule (FIG. 3). As a consequence the amide at 290Asn maintains the
hydrogen bond to 245Asp. In addition, the 0.delta.1 atom of 290Asn can
hydrogen bond to the hydroxyl of 12Ser.
The hydroxyl group of 52Thr, hydrogen bonded to ne2 of 290His in the w.t.
structures, can no longer hydrogen bond to 290Asn. In the mutant the
hydroxyl group of 52Thr is rotated (X.sup.1 torsion) in such a way that it
hydrogen bonds to a water molecule (wildtype X.sup.1 =71.degree., mutant
X.sup.1 =-172.5.degree.). This water molecule present in the mutant
structure is positioned as the 88Met s.delta. atom (distance=0.45.ANG.) in
the wildtype. The introduction of a water molecule at this position, which
forms a hydrogen bond to 52Thr, forces a reorientation of the side chain
of 88Met in the mutant structure as compared to wildtype. In addition the
52Thr side chain hydrogen bonds in the mutant to the main chain hydrogen
of 53phe (d=2.23.ANG.).
The reorientation of the 88Met side chain in H290N necessitates the
movement of 243His. In addition the water molecule (490), bridging 12Ser
to 243His in the wildtype structure, disappears because of steric
hindrance with the methyl group of 52Thr and the new orientation of 88Met.
243His adopts another X angle (in w.t. X.sup.1 =-169.degree. and in mutant
X.sup.1 =-77.degree.) abolishing the hydrogen bond with 215Asn. The space
left by the imidazole of 243His is filed with a water molecule (615) which
hydrogen bonds to the amide of 215Asn (not shown). In addition, this water
molecule hydrogen bonds to a novel water molecule (872) which hydrogen
bonds to main chain hydrogen atom of 244Ile.
Additional evidence for the change in flexibility of the side chains in the
C6 hydroxyl environment is given by the temperature factors for the
residues 52Thr, 88Met and 243His, which is twice as high compared to the
wildtype structure.
As a result of the complex rearrangements caused by the H290N mutation,
three water molecules are solvating the C6 hydroxyl group of the sorbitol.
This exemplifies that replacing, moving or shifting bond water molecules in
the active site can result in desired changes in enzyme activity.
Example 5: Glucose Isomerase with an Improved Substrate Binding
In Table 5 several mutants with improved xylose binding are shown. A25K and
E186Q have a decreased K.sub.m for both xylose and glucose. E186Q has also
a decreased K.sub.m for fructose. Enzymes with high substrate affinity are
preferred under conditions of low substrate concentrations.
TABLE 5
______________________________________
MU-
TANT K(xy) K(gl) K(xy)/Kgl)
K(xy)/WT
K(gl)/WT
______________________________________
E186Q 1.800 57.000 0.032 0.375 0.197
A25K 2.730 207.000 0.013 0.569 0.714
L258K 4.440 327.000 0.014 0.925 1.128
K294R 4.500 308.000 0.015 0.938 1.062
Wild- 4.800 290.000 0.017 1.000 1.000
type
______________________________________
K.sub.m values are expressed in mM. Note that only E186Q and A25K have an
improved affinity for both substrates tested.
Example 6:Mutant with an Improved Specificity for Glucose
The relative specificity is defined as the V.sub.max /K.sub.m ratio for
xylose divided by the V.sub.max /K.sub.m ratio for glucose. Therefore, if
the number is smaller than for the wild-type the relative specificity for
glucose is increased.
In Table 6 mutant glucose isomerases with an increased relative specificity
for glucose are shown. Mutants V135Q, T90SV135Q and T90SV135QN215S have a
largely reduced activity on xylose. Even though in absolute terms the
activity with glucose is reduced, the ratio of V.sub.max /K.sub.m of
glucose over xylose has been improved considerably. Therefore these
mutants have changed their specificity to become a real glucose isomerase
virtually without xylose isomerase activity.
The combination of mutations T90S, V135Q and N215S into a triple mutant
shows that these mutations are additive with respect to the kinetic
parameters (Table 6).
TABLE 6
______________________________________
MUTANT V/K(xy) V/K(gl) V/K(xy)/(gl)
r.spec./WT
______________________________________
T90SV135Q,
0.003 0.040 0.065 0.002
N215S
N215S 0.058 0.018 3.193 0.076
T90SV135Q
0.003 0.007 0.434 0.010
V135Q 0.012 0.005 2.402 0.057
K294Q 0.178 0.031 5.674 0.135
M88SH290N
1.084 0.123 8.846 0.211
V135T 0.547 0.049 11.209 0.267
E186Q 0.428 0.036 11.837 0.282
T90S 2.212 0.182 12.138 0.289
K253RH290N
2.086 0.146 14.053 0.334
H290N 2.474 0.167 14.798 0.352
M88SH243N
1.166 0.065 18.066 0.430
M88S 2.549 0.112 22.768 0.542
E186D 1.216 0.051 23.609 0.562
A25K 2.806 0.109 25.688 0.611
H243N 3.259 0.122 26.661 0.635
K294R 3.067 0.089 34.322 0.817
L258K 5.457 0.135 40.401 0.962
Wildtype 5.042 0.120 42.014 1.000
______________________________________
Example 7: Structural Changes Occurring in the M88SH243N Mutant
Substrate specificity can also be changed by amino acid replacements
resulting in amino acids that show a greater side-chain flexibility.
In the crystal structure of the mutant M88SH243N it is observed that the
243Asn and 52Thr adopt multiple conformations, reflecting the increase of
flexibility in the side chain environment of the C6 hydroxyl environment
of the substrate.
It is also observed that the C.alpha. positions of the adjacent beta barrel
strands containing residues 88Ser, 52Thr, 135Val and 177Arg are displaced
by 0.3 to 0.5.ANG., making the interior of the barrel slightly larger.
The space created by the 88Met to 88Ser mutation is filled with an
additional water molecule. The observed alternate side chain locations and
the slight movement of the barrel allow the introduction of another water
molecule and the movement of a water molecule in the C6 hydroxyl direction
of the substrate.
Example 8:Mutants with Improved Properties in the Presence of Mn.sup.2+
In addition to Mg.sup.2+, Mn.sup.2+ can be used as a bivalent cation
during isomerisation. Although Mn.sup.2+ is not commonly used in
commercial isomerisation processes, its use can be envisaged for
applications in which metal ions can be removed from the product or in
which metal ions are not relevant for the quality of the product.
Mutant E186Q shows an improved catalytic activity towards glucose in the
presence of Mn.sup.2+, see Table 7:
TABLE 7
______________________________________
V.sub.max
(.mu.moles/
min/mg) Km(mM) Conditions
______________________________________
E186Q
Xylose 5.4 3.15 35.degree. C., 1 mM Mn, pH 7.5
Xylose 14.4 7.83 35.degree. C., 2 mM Mn, pH 6.4
Glucose 22.6 387 60.degree. C., 1 mM Mn, pH 7.5
Mn 5.63 0.01 35.degree. C., 100 mM xylose,pH 7.5
Mn 14.5 <0.03 35.degree. C., 200 mM xylose,pH 6.4
Wildtype
Xylose 8.6 13.2 35.degree. C., 1 mM Mn, pH 7.5
Glucose 6.5 1537 60.degree. C., 1 mM Mn, Ph 7.5
Mn 10.2 0.0048 35.degree. C., 100 mM xylose, pH
______________________________________
7.5
The methods used were as in Example 1. The improvement in V.sub.max glucose
is 3- to 4-fold as compared to the wild-type enzyme. The improvement in
K.sub.m glucose is 4-fold. Since 186E is in the vicinity of the UP metal
position (see FIG. 1), it can be envisaged that 186Q is in better concert
with the larger Mn.sup.2+ radius than with the Mg.sup.2+ radius.
Example 9 : Application Testing of the Mutant K253RH290N
Mutant H290N shows an increased activity on glucose as can be seen in Table
4. Table 6 shows that the specificity for glucose is also increased in
this mutant. This mutant was immobilized as described in EP-A-351029
(Example 7 of that application). Application testing of the wildtype and
this mutant glucose isomerase was performed as described in the same
application (Example 8). The stability is indicated by the first order
decay constant (K.sub.d, the lower the decay constant the more stable the
enzyme). Table 8 gives the K.sub.d values for the wildtype and mutant
glucose isomerases.
TABLE 8
______________________________________
Decay constants for wildtype and mutant glucose isomerase,
immobilized on Lewatit
K.sub.d (.times.10.sup.6 sec.sup.-1)
______________________________________
Wildtype 2.5
H290N 3.1
K253R 0.7
H290NK253R 1.6
______________________________________
As can be seen in Table 8, H290N is destabilized as compared with the
wildtype glucose isomerase. K253R was found to stabilize the wildtype
glucose isomerase by a factor larger than three. Combination of H290N with
the stability mutation K253R shows that these characteristics are
additive. Furthermore, it can be seen in Table 4 that the activity of
K253RH290N on glucose is not negatively influenced by the stability
mutation, on the contrary the double mutant shows an even higher activity
on this substrate than mutant H290N. As far as the specificity is
concerned, in Table 6 it can be seen that the K253R mutation does not
substantially influence the specificity of the H290N mutant.
Thus it can be concluded that activity mutants can be stabilized by
introducing mutations that have been shown to stabilize the wildtype
enzyme.
It is to be understood that the above mentioned examples are meant to
demonstrate the concept of the invention and that they are not meant to
limit the scope. In view of this it is clear that combinations of the
above mentioned mutations combined with other mutations leading to altered
characteristics e.g. thermostability, shifted pH optimum or metal binding
are within the scope of the subject invention.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 9
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 394 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
MetSerValGlnA laThrArgGluAspLysPheSerPheGlyLeuTrp
151015
ThrValGlyTrpGlnAlaArgAspAlaPheGlyAspAlaThrArgThr
202530
AlaLeuAspProValGluAlaValHisLysLeuAlaGluIleGlyAla
354045
TyrGlyIleThrPheH isAspAspAspLeuValProPheGlySerAsp
505560
AlaGlnThrArgAspGlyIleIleAlaGlyPheLysLysAlaLeuAsp
6570 7580
GluThrGlyLeuIleValProMetValThrThrAsnLeuPheThrHis
859095
ProValPheLysAspG lyGlyPheThrSerAsnAspArgSerValArg
100105110
ArgTyrAlaIleArgLysValLeuArgGlnMetAspLeuGlyAlaGlu
115 120125
LeuGlyAlaLysThrLeuValLeuTrpGlyGlyArgGluGlyAlaGlu
130135140
TyrAspSerAlaLysAspValSerAl aAlaLeuAspArgTyrArgGlu
145150155160
AlaLeuAsnLeuLeuAlaGlnTyrSerGluAspArgGlyTyrGlyLeu
165 170175
ArgPheAlaIleGluProLysProAsnGluProArgGlyAspIleLeu
180185190
LeuProThrAlaGlyHis AlaIleAlaPheValGlnGluLeuGluArg
195200205
ProGluLeuPheGlyIleAsnProGluThrGlyHisGluGlnMetSer
210 215220
AsnLeuAsnPheThrGlnGlyIleAlaGlnAlaLeuTrpHisLysLys
225230235240
LeuPheHisIleAspLeuAsn GlyGlnHisGlyProLysPheAspGln
245250255
AspLeuValPheGlyHisGlyAspLeuLeuAsnAlaPheSerLeuVal
260 265270
AspLeuLeuGluAsnGlyProAspGlyAlaProAlaTyrAspGlyPro
275280285
ArgHisPheAspTyrLysProS erArgThrGluAspTyrAspGlyVal
290295300
TrpGluSerAlaLysAlaAsnIleArgMetTyrLeuLeuLeuLysGlu
305310 315320
ArgAlaLysAlaPheArgAlaAspProGluValGlnGluAlaLeuAla
325330335
AlaSerLysValAlaGluLe uLysThrProThrLeuAsnProGlyGlu
340345350
GlyTyrAlaGluLeuLeuAlaAspArgSerAlaPheGluAspTyrAsp
355 360365
AlaAspAlaValGlyAlaLysGlyPheGlyPheValLysLeuAsnGln
370375380
LeuAlaIleGluHisLeuLeuGlyAlaArg
385390
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 394 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
MetSerLeuGlnAlaThrProAspAspLysPheSerPh eGlyLeuTrp
151015
ThrValGlyTrpGlnAlaArgAspAlaPheGlyAspAlaThrArgPro
2025 30
ValLeuAspProIleGluAlaValHisLysLeuAlaGluIleGlyAla
354045
TyrGlyValThrPheHisAspAspAspLeuValProPheGl yAlaAsp
505560
AlaAlaThrArgAspGlyIleValAlaGlyPheSerLysAlaLeuAsp
657075 80
GluThrGlyLeuIleValProMetValThrThrAsnLeuPheThrHis
859095
ProValPheLysAspGlyGlyPheThrSerAsnAspArgSe rValArg
100105110
ArgTyrAlaIleArgLysValLeuArgGlnMetAspLeuGlyAlaGlu
115120125
LeuGlyAlaLysThrLeuValLeuTrpGlyGlyArgGluGlyAlaGlu
130135140
TyrAspSerAlaLysAspValGlyAlaAlaLeuAspArgTyrArgGlu
145150155160
AlaLeuAsnLeuLeuAlaGlnTyrSerGluAspGlnGlyTyrGlyLeu
165170 175
ProPheAlaIleGluProLysProAsnGluProArgGlyAspIleLeu
180185190
LeuProThrAlaGlyHisAlaIleAlaPheValGlnGluLeu GluArg
195200205
ProGluLeuPheGlyIleAsnProGluThrGlyHisGluGlnMetSer
210215220
As nLeuAsnPheThrGlnGlyIleAlaGlnAlaLeuTrpHisLysLys
225230235240
LeuPheHisIleAspLeuAsnGlyGlnHisGlyProLysPheAspG ln
245250255
AspLeuValPheGlyHisGlyAspLeuLeuAsnAlaPheSerLeuVal
26026527 0
AspLeuLeuGluAsnGlyProAspGlyGlyProAlaTyrAspGlyPro
275280285
ArgHisPheAspTyrLysProSerArgThrGluAspPheAspGlyVa l
290295300
TrpGluSerAlaLysAspAsnIleArgMetTyrLeuLeuLeuLysGlu
305310315320
ArgAlaLysAlaPheArgAlaAspProGluValGlnAlaAlaLeuAla
325330335
GluSerLysValAspGluLeuArgThrProThrLeuAsnProGly Glu
340345350
ThrTyrAlaAspLeuLeuAlaAspArgSerAlaPheGluAspTyrAsp
355360365
AlaAspAlaValGlyAlaLysGlyTyrGlyPheValLysLeuAsnGln
370375380
LeuAlaIleAspHisLeuLeuGlyAlaArg
385390
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 387 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
MetAsnTyrGlnProThrProGluAspArgPheThrPheGlyLeuTrp
1 51015
ThrValGlyTrpGlnGlyArgAspProPheGlyAspAlaThrArgGln
202530
AlaLeuAspPro AlaGluSerValArgArgLeuSerGluLeuGlyAla
354045
TyrGlyValThrPheHisAspAspAspLeuIleProPheGlySerSer
50 5560
AspThrGluArgGluSerHisIleLysArgPheArgGlnAlaLeuAsp
65707580
AlaThrGlyMetLysVal ProMetAlaThrThrAsnLeuPheThrHis
859095
ProValPheLysAspGlyAlaPheThrAlaAsnAspArgAspValArg
100 105110
ArgTyrAlaLeuArgLysThrIleArgAsnIleAspLeuAlaValGlu
115120125
LeuGlyAlaSerValTyrV alAlaTrpGlyGlyArgGluGlyAlaGlu
130135140
SerGlyAlaAlaLysAspValArgAspAlaLeuAspArgMetLysGlu
145150 155160
AlaPheAspLeuLeuGlyGluTyrValThrGluGlnGlyTyrAspLeu
165170175
LysPheAlaIleGluPr oLysProAsnGluProArgGlyAspIleLeu
180185190
LeuProThrValGlyHisAlaLeuAlaPheIleGluArgLeuGluArg
195 200205
ProGluLeuTyrGlyValAsnProGluValGlyHisGluGlnMetAla
210215220
GlyLeuAsnPheProHisGlyIleAla GlnAlaLeuTrpAlaGlyLys
225230235240
LeuPheHisIleAspLeuAsnGlyGlnSerGlyIleLysTyrAspGln
245 250255
AspLeuArgPheGlyAlaGlyAspLeuArgAlaAlaPheTrpLeuVal
260265270
AspLeuLeuGluArgAla GlyTyrAlaGlyProArgHisPheAspPhe
275280285
LysProProArgThrGluAspPheAspGlyValTrpAlaSerAlaAla
2902 95300
GlyCysMetArgAsnTyrLeuIleLeuLysAspArgAlaAlaAlaPhe
305310315320
ArgAlaAspProGlnValGlnG luAlaLeuAlaAlaAlaArgLeuAsp
325330335
GluLeuAlaArgProThrAlaGluAspGlyLeuAlaAlaLeuLeuAla
340 345350
AspArgSerAlaTyrAspThrPheAspValAspAlaAlaAlaAlaArg
355360365
GlyMetAlaPheGluHisLeuAs pGlnLeuAlaMetAspHisLeuLeu
370375380
GlyAlaArg
385
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 388 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
MetSerPheGlnProThrProGluAspArgPheThrPheGlyLeuTrp
151015
ThrValGlyTrpGlnGlyArg AspProPheGlyAspAlaThrArgPro
202530
AlaLeuAspProValGluThrValGlnArgLeuAlaGluLeuGlyAla
35 4045
TyrGlyValThrPheHisAspAspAspLeuIleProPheGlySerSer
505560
AspThrGluArgGluSerHisIleLysArgPhe ArgGlnAlaLeuAsp
65707580
AlaThrGlyMetThrValProMetAlaThrThrAsnLeuPheThrHis
85 9095
ProValPheLysAspGlyGlyPheThrAlaAsnAspArgAspValArg
100105110
ArgTyrAlaLeuArgLysThrIleGly AsnIleAspLeuAlaAlaGlu
115120125
LeuGlyAlaLysThrTyrValAlaTrpGlyGlyArgGluGlyAlaGlu
130135 140
SerGlyGlyAlaLysAspValArgAspAlaLeuAspArgMetLysGlu
145150155160
AlaPheAspLeuLeuGlyGluTyrValThr AlaGlnGlyTyrAspLeu
165170175
ArgPheAlaIleGluProLysProAsnGluProArgGlyAspIleLeu
1801 85190
LeuProThrValGlyHisAlaLeuAlaPheIleGluArgLeuGluArg
195200205
ProGluLeuTyrGlyValAsnProGluValG lyHisGluGlnMetAla
210215220
GlyLeuAsnPheProHisGlyIleAlaGlnAlaLeuTrpAlaGlyLys
225230235 240
LeuPheHisIleAspLeuAsnGlyGlnSerGlyIleLysTyrAspGln
245250255
AspLeuArgPheGlyAlaGlyAspLeuAr gAlaAlaPheTrpLeuVal
260265270
AspLeuLeuGluThrAlaGlyTyrGluGlyProArgHisPheAspPhe
275280 285
LysProProArgThrGluAspPheAspGlyValTrpAlaSerAlaAla
290295300
GlyCysMetArgAsnTyrLeuIleLeuLysAspArgAla AlaAlaPhe
305310315320
ArgAlaAspProGluValGlnGluAlaLeuArgAlaAlaArgLeuAsp
325330 335
GlnLeuAlaGlnProThrAlaAlaAspGlyLeuAspAlaLeuLeuAla
340345350
AspArgAlaAlaPheGluAspPheAspVal AspAlaAlaAlaAlaArg
355360365
GlyMetAlaPheGluHisLeuAspGlnLeuAlaMetAspHisLeuLeu
370375 380
GlyAlaArgGly
385
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 347 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
MetSerTyrGlnProThrProGluAspArgP heSerPheGlyLeuTrp
151015
ThrValGlyTrpGlnGlyArgAspProPheGlyAspAlaThrArgArg
2025 30
ProLeuAspProValGlyThrValGlnArgLeuAlaGluLeuGlyAla
354045
TyrGlyValThrPheHisAspAspAspLeuIleP roPheGlyAlaSer
505560
GluAlaGluArgGluAlaHisValLysArgPheArgGlnAlaLeuAsp
657075 80
AlaThrGlyMetThrValProMetAlaThrThrAsnLeuPheThrHis
859095
ProValPheLysAspGlyAlaPheThrAlaAsnA spArgAspValArg
100105110
ArgTyrAlaLeuArgLysThrIleArgAsnIleAspLeuAlaValGlu
115120 125
LeuGlyAlaArgThrTyrValAlaTrpGlyGlyArgGluGlyAlaGlu
130135140
SerGlyAlaAlaLysAspValArgAlaAlaLeuAspArgMetLy sGlu
145150155160
AlaPheAspLeuLeuGlyGluTyrValThrSerGlnGlyTyrAspIle
165170 175
ArgPheAlaIleGluProLysProAsnGluProArgGlyAspIleLeu
180185190
LeuProThrValGlyHisAlaLeuAlaPheIleGlu ArgLeuGluArg
195200205
ProGluLeuPheGlyValAsnProGluValGlyHisGluGlnMetAla
210215220
GlyLeuAsnPheProHisGlyIleAlaGlnAlaLeuTrpAlaGlyLys
225230235240
LeuPheHisIleAspLeuAsnGlyGlnSerGlyIleLys TyrAspGln
245250255
AspLeuArgPheGlyAlaGlyAspLeuArgAlaAlaPheTrpLeuVal
260265 270
AspLeuLeuGluSerSerGlyTyrAspGlyProArgHisPheAspPhe
275280285
LysProProArgThrGluAspLeuAspGlyValTrpAlaS erAlaAla
290295300
GlyCysMetArgAsnTyrLeuIleLeuLysGluArgSerAlaAlaPhe
305310315 320
ArgAlaAspProGluValGlnGluAlaLeuArgAlaSerArgLeuAsp
325330335
GlnLeuAlaGlnProThrAlaAlaAspGlyLeu
340345
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 395 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
MetSerValGlnProThrProAlaAspHisPheT hrPheGlyLeuTrp
151015
ThrValGlyTrpThrGlyAlaAspProPheGlyValAlaThrArgLys
2025 30
AsnLeuAspProValGluAlaValHisLysLeuAlaGluLeuGlyAla
354045
TyrGlyIleThrPheHisAspAsnAspLeuIleProP heAspAlaThr
505560
GluAlaGluArgGluLysIleLeuGlyAspPheAsnGlnAlaLeuLys
657075 80
AspThrGlyLeuLysValProMetValThrThrAsnLeuPheSerHis
859095
ProValPheLysAspGlyGlyPheThrSerAsnAspA rgSerIleArg
100105110
ArgPheAlaLeuAlaLysValLeuHisAsnIleAspLeuAlaAlaGlu
115120 125
MetGlyAlaGluThrPheValMetTrpGlyGlyArgGluGlySerGlu
130135140
TyrAspGlySerLysAspLeuAlaAlaAlaLeuAspArgMetArgGl u
145150155160
GlyValAspThrAlaAlaGlyTyrIleLysAspLysGlyTyrAsnLeu
165170 175
ArgIleAlaLeuGluProLysProAsnGluProArgGlyAspIlePhe
180185190
LeuProThrValGlyHisGlyLeuAlaPheIleGluGln LeuGluHis
195200205
GlyAspIleValGlyLeuAsnProGluThrGlyHisGluGlnMetAla
210215220
GlyLeuAsnPheThrHisGlyIleAlaGlnAlaLeuTrpAlaGluLys
225230235240
LeuPheHisIleAspLeuAsnGlyGlnArgGlyIleLysTyr AspGln
245250255
AspLeuValPheGlyHisGlyAspLeuThrSerAlaPhePheThrVal
260265 270
AspLeuLeuGluAsnGlyPheProAsnGlyGlyProLysTyrThrGly
275280285
ProArgHisPheAspTyrLysProSerArgThrAspGlyTyrA spGly
290295300
ValTrpAspSerAlaLysAlaAsnMetSerMetTyrLeuLeuLeuLys
30531031532 0
GluArgAlaLeuAlaPheArgAlaAspProGluValGlnGluAlaMet
325330335
LysThrSerGlyValPheGluLeuGlyGluThrThrLeuAs nAlaGly
340345350
GluSerAlaAlaAspLeuMetAsnAspSerAlaSerPheAlaGlyPhe
355360365
AspAlaGluAlaAlaAlaGluArgAsnPheAlaPheIleArgLeuAsn
370375380
GlnLeuAlaIleGluHisLeuLeuGlySerArg
385 390395
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 440 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
MetAlaGlnSerHisSerSerSerValAsnTyrPheGlySe rValAsn
151015
LysValValPheGluGlyLysAlaSerThrAsnProLeuAlaPheLys
2025 30
TyrTyrAsnProGlnGluValIleGlyGlyLysThrMetLysGluHis
354045
LeuArgPheSerIleAlaTyrTrpHisThrPheThrAlaAspGl yThr
505560
AspValPheGlyAlaAlaThrMetGlnArgProTrpAspHisTyrLys
65707580
GlyMetAspLeuAlaArgAlaArgValGluAlaAlaPheGluMetPhe
859095
GluLysLeuAspAlaProPhePheAlaPheHisAspArgAspIl eAla
100105110
ProGluGlySerThrLeuLysGluThrAsnGlnAsnLeuAspIleIle
115120125
ValGlyMetIleLysAspTyrMetArgAspSerAsnValLysLeuLeu
130135140
TrpAsnThrAlaAsnMetPheThrAsnProArgPheValHisGlyAla
1 45150155160
AlaThrSerCysAsnAlaAspValPheAlaTyrAlaAlaAlaGlnVal
165170175
LysLysGlyLeuGluThrAlaLysGluLeuGlyAlaGluAsnTyrVal
180185190
PheTrpGlyGlyArgGluGlyTyrGluThrLeuLeuAsnThrAsp Leu
195200205
LysPheGluLeuAspAsnLeuAlaArgPheMetHisMetAlaValAsp
210215220
TyrAl aLysGluIleGluTyrThrGlyGlnPheLeuIleGluProLys
225230235240
ProLysGluProThrThrHisGlnTyrAspThrAspAlaAlaThrThr
245250255
IleAlaPheLeuLysGlnTyrGlyLeuAspAsnHisPheLysLeuAsn
260265270
LeuGluAlaAsnHisAlaThrLeuAlaGlyHisThrPheGluHisGlu
275280285
LeuArgMetAlaArgValHisGlyLeuLeuGlySerValAspAlaAsn
290295300
GlnGlyHisProLeuLeuGlyTrpAspThrAspGluPheProThrAsp
305310315320
LeuTyrSerThrThrLeuAlaMetTyrGluIleLeuGlnAsnGlyGly
325330335
LeuGlySerGlyGlyLeuAsnPheAspAlaLysValArgArgSerSer
340345350
PheGluProAspAspLeuValTyrAlaHisIleAlaGlyMetAspAla
355360365
P heAlaArgGlyLeuLysValAlaHisLysLeuIleGluAspArgVal
370375380
PheGluAspValIleGlnHisArgTyrArgSerPheThrGluGlyIle
385 390395400
GlyLeuGluIleThrGluGlyArgAlaAsnPheHisThrLeuGluGln
405410415
TyrAlaLeuAsnAsnLysThrIleLysAsnGluSerGlyArgGlnGlu
420425430
ArgLeuLysProIleLeuAsnGln
435 440
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 440 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
MetGlnAlaTyrPheAspGlnLeuAspArgValArgTyrGluGlySer
1 51015
LysSerSerAsnProLeuAlaPheArgHisTyrAsnProAspGluLeu
202530
ValLeu GlyLysArgMetGluGluHisLeuArgPheAlaAlaCysTyr
354045
TrpHisThrPheCysTrpAsnGlyAlaAspMetPheGlyValGlyAla
50 5560
PheAsnArgProTrpGlnGlnProGlyGluAlaLeuAlaLeuAlaLys
65707580
ArgLysAlaAsp ValAlaPheGluPhePheHisLysLeuHisValPro
859095
PheTyrCysPheHisAspValAspValSerProGluGlyAlaSerLeu
100105110
LysGluTyrIleAsnAsnPheAlaGlnMetValAspValLeuAlaGly
115120125
LysGlnGluGluSe rGlyValLysLeuLeuTrpGlyThrAlaAsnCys
130135140
PheThrAsnProArgTyrGlyAlaGlyAlaAlaThrAsnProAspPro
145150 155160
GluValPheSerTrpAlaAlaThrGlnValValThrAlaMetGluAla
165170175
ThrHisLysLeu GlyGlyGluAsnTyrValLeuTrpGlyGlyArgGlu
180185190
GlyTyrGluThrLeuLeuAsnThrAspLeuArgGlnGluArgGluGln
195 200205
LeuGlyArgPheMetGlnMetValValGluHisLysHisLysIleGly
210215220
PheGlnGlyThrLeuLeuIle GluProLysProGlnGluProThrLys
225230235240
HisGlnTyrAspTyrAspAlaAlaThrValTyrGlyPheLeuLysGln
2 45250255
PheGlyLeuGluLysGluIleLysLeuAsnIleGluAlaAsnHisAla
260265270
ThrLeuAlaGlyH isSerPheHisHisGluIleAlaThrAlaIleAla
275280285
LeuGlyLeuPheGlySerValAspAlaAsnArgGlyAspAlaGlnLeu
290 295300
GlyTrpAspThrAspGlnPheProAsnSerValGluGluAsnAlaLeu
305310315320
ValMetTyrGluIleLe uLysAlaGlyGlyPheThrThrGlyGlyLeu
325330335
AsnPheAspAlaLysValArgArgGlnSerThrAspLysTyrAspLeu
340 345350
PheTyrGlyHisIleGlyAlaMetAspThrMetAlaLeuAlaLeuLys
355360365
IleAlaAlaArgMetIle GluAspGlyGluLeuAspLysArgIleAla
370375380
GlnArgTyrSerGlyTrpAsnSerGluLeuGlyGlnGlnIleLeuLys
385390 395400
GlyGlnMetSerLeuAlaAspLeuAlaLysTyrAlaGlnGluHisHis
405410415
LeuSerProValHis GlnSerGlyArgGlnGluGlnLeuGluAsnLeu
420425430
ValAsnHisTyrLeuPheAspLys
435440
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 439 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
MetAlaTyrPheAsnAspIleAlaProIleLysTyrGluGlyThrLys
15 1015
ThrLysAsnMetPheAlaPheArgHisTyrAsnProGluGluValVal
202530
AlaGlyLysThrMetGluGluGl nLeuHisPheAlaLeuAlaPheTrp
354045
HisThrIleThrMetAspGlySerAspProPheGlyGlyAlaThrMet
5055 60
GluArgProTrpAspLeuGluGlyGlySerGluLeuAspArgAlaHis
65707580
ArgArgValAspAlaPhePheGluIleAl aGluLysLeuGlyValLys
859095
TyrTyrCysPheHisAspIleAspIleAlaProThrGlyAsnSerLeu
100 105110
LysGluPheTyrAlaAsnLeuAspGluIleThrAspHisLeuLeuGlu
115120125
LysGlnLysAlaThrGlyIleLysLeuLeu TrpAsnThrAlaAsnMet
130135140
PheSerAsnProArgTyrMetAsnGlyValSerThrSerAsnArgAla
145150155 160
GluValPheAlaTyrGlyAlaAlaGlnValLysLysGlyLeuGluLeu
165170175
SerLysLysLeuGlyGlyGluAsnTyrV alPheTrpGlyGlyArgGlu
180185190
GlyTyrGluSerLeuLeuAsnThrAspMetGlyLeuGluMetAspHis
195200 205
MetAlaLysPhePheHisLeuAlaIleAspTyrAlaLysSerIleAsn
210215220
HisLeuProIlePheLeuIleGluProLysProLysGl uProMetThr
225230235240
HisGlnTyrAspPheAspSerAlaThrAlaLeuAlaPheLeuGlnLys
245250 255
TyrAspLeuAspLysTyrPheLysLeuAsnLeuGluThrAsnHisAla
260265270
TrpLeuAlaGlyHisThrPheGluHisGlu LeuAsnThrAlaArgThr
275280285
PheAsnAlaLeuGlySerIleAspAlaAsnGlnGlyAsnTyrLeuLeu
290295 300
GlyTrpAspThrAspGluPheProThrLeuValIleAspIleThrLeu
305310315320
AlaMetHisGlnIleLeuLeuAsnGlyGlyLeu GlyLysGlyGlyIle
325330335
AsnPheAspAlaLysValArgArgThrSerPheLysAlaGluAspLeu
340345 350
IleLeuAlaHisIleAlaGlyMetAspThrTyrAlaArgAlaLeuLys
355360365
GlyAlaAlaAlaIleIleGluAspLysPheLeuS erAspIleValAsp
370375380
GluArgTyrSerSerTyrArgAsnThrGluValGlyGlnSerIleGlu
385390395 400
AsnGlyThrAlaThrPheGluSerLeuAlaAlaPheAlaLeuGluTyr
405410415
GlyAspAspIleGluLeuAspSerAsnHisLe uGluTyrIleLysSer
420425430
ValLeuAsnAspTyrLeuVal
435
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